Abstract
Glycosylasparaginase is an N-terminal nucleophile hydrolase and is activated by intramolecular autoproteolytic processing. This cis-autoproteolysis possesses unique kinetics characterized by a reversible N-O acyl rearrangement step in the processing. Arg-180 and Asp-183, involved in binding of the substrate in the mature enzyme, are also involved in binding of free amino acids in the partially formed substrate pocket on certain mutant precursors. This binding site is sequestered in the wild-type precursor. Binding of free amino acids on mutant precursors can either inhibit or accelerate their processing, depending on the individual mutants and amino acids. The polypeptide sequence at the processing site, which is highly conserved, adopts a special conformation. Asp-151 is essential for maintaining this conformation, possibly by anchoring its side chain into the partially formed substrate pocket through interaction with Arg-180. The reactive nucleophile Thr-152 is activated not only by deprotonation by His-150 but also by interaction with Thr-170, suggesting a His-Thr-Thr active triad for the autoproteolysis.
Highlights
Glycosylasparaginase hydrolyzes the -N-glycosidic bond between asparagine and N-acetylglucosamine of asparaginelinked glycans [1]
Since Ntn-enzymes have in common an unusual fold and the active N-terminal nucleophile is most often generated by autoproteolytic processing [5], we suggested that the cis-autoproteolysis model may with some variations, be held for processing of other Ntn-enzymes
We fused the genes of the E. coli maltose-binding protein (MBP) and glycosylasparaginase, termed MG, and expressed the gene fusion in E. coli
Summary
By studying in vitro autoprocessing of mutant precursors, we demonstrate that the activation of glycosylasparaginase follows the kinetics of cis-autoproteolysis. Site-directed mutagenesis and in vitro activation of purified precursors show that two residues, Arg-180 and Asp-183, which are involved in binding of substrate in the mature enzyme, are involved in binding of free amino acids on mutant precursors. Kinetic Analysis—The cis-autoproteolytic processing of the glycosylasparaginase proenzyme is described by the following reaction, k1. We have used k/k or (NH2OH)/(H2O) to estimate the relative ester hydrolysis rates, where (NH2OH) and (H2O) are the half processing times with and without 0.25 M NH2OH. The inhibition constant Ki of amino acid is determined as described previously [9]
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